Vol. 69, No. 3, 1976
SOLUBILIZATION
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
AND PARTIAL
PURIFICATION
OF ALANINE CARRIER
FROM MEMBRANES OF A THERMOPHILIC BACTERIUM AND ITS RECONSTITUTION INTO FUNCTIONAL VESICLES Hajime
Hirata, Nobuhito Sone, Masasuke Yoshida and Yasuo Kagawa Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken, Japan 329-04
Received
January
27,1976 SUMMARY
An alanine transport carrier was solubilized from membranes of the thermophilic bacterium PS3 with cholate-deoxycholate mixture. It was then partially purified by diethyl aminoethyl cellulose column chromatography and gel filtration. For assay of alanine carrier activity it was reconstituted into vesicles with P-lipids and the transport energy was supplied as a membrane potential introduced by K+-diffusion via valinomycin. The partially purified carrier had no ATPase or NADH dehydrogenase activity. Active transport of alanine driven by the membrane potential was completely abolished by an uncoupler.
Bacterial tensively
membrane transport
using
isolated
showed that
coupling
interpreted
by Mitchell's
obtain
bacterial riers
of metabolic
no transport
are too studies It
branes
unstable
various
the
drugs,
a dicyclohexyl from this
that
thermophilic organic
solvents
using
0 1976 by Academic Press, Inc. of reproduction in any form reserved.
However,
to
required. from
known transport
be obtained
PS3, which
car-
Thus for
from the
are resistant (6).
ATPase complex
and noninonic 665
be
required.
and detergents
sensitive ionic
is
could
bacterium
carbodiimide
bacterium
this
(2 - 5).
from membranes.
material
results
been isolated all
in-
can best
system is
has yet
to be solubilized
seemed possible of
theory
may be because
a more stable
Recent
to transport
a much purer carrier
This
has been studied (1).
energy
chemiosmotic results
membranes.
further
Copyright All rights
membrane vesicles
more convincing
Unfortunately,
of nutrients
Indeed
mem-
to recently
was purified
detergents
(7).
Vol. 69, No. 3, 1976
This
BIOCHEMICAL
communication
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
describes
the
purification
of an alanine
carrier
constitution
with
into
port
P-lipids
solubilization
from this vesicles
and partial
bacterium
capable
of
and its
active
re-
trans-
of alanine.
MATERIALS AND METHODS Materia& The thermophilic bacterium PS3 (kindly donated by Dr. T. Oshima) and preparation of membranes were described previously (6). Soybean P-lipids (asolectin) were obtained from Associated Concentrates, Woodside, New York, and partially purified as described previously (7). The preparation of lipids of PS3 was was purchased also as described previously (7). L-[U- 14C]alanine from Daiichi Radiochemicals. Cholic acid was recrystallized as described by Kagawa (8). Other compounds were commercial products. P-Lipid-detergent Mixture P-Lipids (10 mg) were suspended in 2 ml of 40 mM Tris-SO4 (pH 8) containing 2% Na-cholate, 1% Na-deoxycholate and 1 mM dithiothreitol, and sonicated in a Tomy probe sonic oscillator, Model UR-150P, at 20 KHz and 150 watts in an ice bath for 5 min. Soihbilization and Partial Purification of Alanine Carrier Membranes (1 g of protein) were suspended in a solution containing Na-cholate, 2%; Na-deoxycholate, 1%; NapSOt,, 0.2 M; Tris-SOI, (pH 8), 30 mM; and dithiothreitol, 0.5 mM in a final volume of 100 ml. The mixture was sonicated in an ice bath for 10 min and then centrifuged at 140,000 x g for 1 hr. The supernatant was concentrated to approximately 20 ml in a Diaflo apparatus with a UMlO filter and dialyzed against 2 liters of 50 mM Tris-SOI, (pH 8) containing 0.25 mM dithiothreitol for 17 hrs at room temperature. The contents in the dialysis bag were then centrifuged at 140,000 x g for 1 hr and the resulting precipitate was suspended in 50 mM Tris-SO,+ (pH 8). This preparation is named CDE-P. CDE-P was treated with Triton X-100 (2% final concentration) in the presence of 0.2 M NapSOb, sonicated for 1 min and centrifuged at 140,000 x g for 1 hr. The supernatant was diluted 20-fold with distilled water and applied on a DEAE cellulose column equilibrated with 25 mM Tris-SO,, (pH 8) containing 0.1% Triton X-100 (Tris-Triton). The column was washed with 10 bed volume of Tris-Triton, and then eluted with Tris-Triton containing 50 mM NazS04. Fractions with UV-absorption were combined and mixed with Na-cholate at a final concentration of 1%. Then 210 mg of solid ammonium sulfate were added. The precipitate was collected by centrifugation (20,000 x g, 10 min) and suspended in 50 mM Tris-SOI, (pH 8) (DE-l). DE-1 was purified further by gel For this it was dissolved in a minimal volume of 2% filtration. Triton X-100 and applied on a Sepharose 6B column equilibrated with Tris-Triton. The column was eluted with the same buffer and the eluate with UV-absorption (one broad peak) was collected and divided into three fractions (Fr. 1, 2 and 3). Reconstitution of Vesicles and &-Loading Samples of the preparations obtained at each step (10 to 100 pg protein) were added to the P-lipid-detergent mixture (2 ml) described above and sonicated for Then the mixtures were dialyzed against 50 mM Tris-SOQ 30 sec. (pH 8) containing 2.5 mM MgSOs, 0.2 mM EDTA and 0.25 mM dithiothreiThe reconstituted vesicles were incuto1 for 17 to 20 hrs at 40". bated in 0.5 M K-phosphate buffer (pH 8) for 30 min at 55O and then
666
BIOCHEMICAL
Vol. 69, No. 3,1976
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
transferred to an ice bath. Then MgSO4 was added to a final concentration of 10 mM and incubation was continued for 30 min in the ice bath. The reconstituted-Kf-loaded vesicles were collected by centrifugation at 140,000 x g for 60 min, washed with 0.4 M sucrose containing 10 mM MgSO1, and suspended in 0.4 M sucrose-10 mM MqSO,+. Assay of ALanine Carrier Activity Alanine carrier was defined as protein(s) catalyzing active transport of radioactive alanine into the reconstituted vesicles depending on an artificial membrane potential (negative inside). The reconstituted-K+ -loaded vesicles were suspended in 1 ml of solution containing final concentrations of 0.1 M Tris-maleate, pH 7.0, 0.013 M MqSO4, 0.28 M sucrose, and 10 PM L-[U-14C]alanine, (1 vCi/ml). After incubation for 5 min at 400, 2 ug of valinomycin in 2 ~1 of methanol were added. At intervals 0.1 ml samples were filtered through membrane filters (Sartorius, 0.45 pm pore size) and washed with 0.4 M sucrose-10 mM MqS04. The filters were then rapidly removed, dried and their radioactivities were assayed in a gas-flow counter (Aloka LBC-451). Protein was determined as described by Lowry et al. (9).
RESULTS AND DISCUSSION A transport catalyzing
movement of
tration
gradient
simplified
in other
alanine
uptake
artificial
nutrient
carrier
a metabolic
form.
(manuscript
for
coli
assay of
of
energy.
similar In this
carrier
must be
work
PS3 was driven
by K+ diffusion
in preparation)
alanine
of
in a
energy
In our previous
introduced
(4).
a concen-
However,
protein(s),
membrane vesicles
vesicles
as protein
a membrane against
only
by isolated
Esckerickia
across sort
containing than
must be determined
on some
membrane potential
valinomycin
method
protein(s)
dependent
system
supplied
with
carrier
to that
mediated
after
by
achieved
work we applied
activity
by an
this
reconstitution
of vesicles. Figure vesicles
reconstituted
soluble tuted
1 illustrates
proteins. from
alanine from the
which
2.
On addition
reached
insoluble
on alanine
uptakes
from cholate-deoxycholate of valinomycin,
the detergent-soluble
The validity in Fig.
results
a maximum in
proteins of
The initial
proteins
this
of
alanine
667
vesicles
Vesicles active
assay method is rate
soluble
showed rapid
2 min.
showed less
by K+-loaded
reconstiuptake
of
reconstituted
uptake.
confirmed
uptake
and in-
by the
results
was proportional
to
Vol. 69, No. 3, 1976
BIOCHEMICAL
-2
AND BIOPHYSICAL
5
0 TIM
Fig.
Fig.
1
2
:
:
RESEARCH COMMUNICATIONS
7
(MIN)
+ Alanine uptake by reconstituted, K -loaded vesicles. Vesicles containing the detergent-soluble ( 0) or -insoluble ( 0) proteins were reconstituted and loaded The with K-phosphate (pH 8) as described in the text. reaction mixture (1 ml) contained final concentrations of 0.1 M Tris-maleate, pH 7.0, 0.013 M MgS04, 0.28 M and 10 UM L-[U-14C]alanine, (1 uCi/ml). After sucrose, incubation for 5 min at 40" (arrow) 2 pg of valinomycin in 2 ~1 of methanol were added. At intervals, 0.1 ml aliquots were filtered and washed.
Alanine uptake by reconstituted-K+-loaded besicles as a function of amount of detergent-soluble proteins added. Reconstituted, K+-loaded vesicles containing various amounts of detergent-soluble proteins and a fixed amount of PS3 P-lipids (10 mg) were prepared as described in the text. The initial velocities ( 0) and maximal uptakes (0) were calculated from the values observed 15 set and 2.5 min after addition of valinomycin.
668
Vol. 69, No. 3, 1976
the
BIOCHEMICAL
amount of protein
ed to a fixed
(CDE-P),
hand,
explained
by supposing
tended
to both
carrier
over
amount of P-lipids
the other
tional
AND BIOPHYSICAL
the
protein(s)
the
range
to reach
a definite
that
initial
size
of the
and the
rate
population
level
was related
thus
reached
a definite
level
when most of
by these
on an artificial blocked
containing in the vesi-
former
only
the vesicles
and
contain
at
protein(s).
The uptake
ly
be
was propor-
protein(s) to the
on
could
of uptake
amount of carrier
the maximal
level,
This
of vesicles
while
one carrier
20 to 600 i-19, add-
level.
cles,
least
of
The maximal
(10 mg).
the
RESEARCH COMMUNICATIONS
reconstituted
vesicles,
membrane potential
by the uncoupler,
phenyl-hydrazone,
but
(negative
which inside),
carbonylcyanide
was dependent was complete-
p-trifluoromethoxy-
not by dicyclohexyl
carbodiimide
(data
not
shown). Various P-lipids, rial
P-lipid
soy bean P-lipids,
strain
phosphatidyl
with
alanine
Thus specific of the
carrier
phosphatidyl
saturated
fatty
only
are required
acyl
groups
not
vesishown).
reconstitution
of PS3 P-lipids
glycerol
of which
formed (data
functional
phosphatidyl
bacte-
and synthetic
activity
for
PS3
thermophilic
PS3 P-lipids
The main components
ethanolamine,
and the
(111,
from other
showing uptake
protein.
including
HB8, ATCC 27634,
glycerol;
carrier
P-lipids
were tested,
those
Thermus thermophilus
dipalmitoyl cles
preparations
are
and cardiolipin
are almost
completely
(7).
The alanine
was purified
as described
in the
AND METHODS" and results
of
purification
are summarized
in Table
of extraction
I.
carrier
The process
cholate-deoxycholate 2.4-fold. lulose
mixture
The alanine column with
carrier
rather
a typical
increased activity
a low salt
669
"MATERIALS
of the membranes with the
specific
was eluted concentration,
activity from a DEAE celsometimes
as
low
Vol. 69, No. 3, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Table Purification
of Alanine
Membrane vesicles Cholate-DOC ext. Triton
ext.
Triton DEAE
SP. Act. n moles/min*mg
1,096 96.3
0.76 1.85
sup
DEAE 200 mM Na2S04 Sepharose Fr. 1
as 25 mM Na2S04. but
small
tion
alanine
specific
step
procedures
16.0 0.65
0.77 9.93
0.64 0.18
5.88 1.84
were still
1 of Table
I.
must be between
of the
it
0.50 5.12
final
contained
are required
present
On Sepharose
membranes and the that
1.89
37.8 2.5
removed ATPase and NADH dehydrogenase
shown).
in Fr.
carrier
activity
original
suggesting tive
not
was found
of the
the
(data
49.7
2 3
amounts of cytochromes
eluted
activity
This
Fr. Fr.
Carrier
Protein hg)
ext. ppt 50 mM Na2S04
Sepharose Sepharose
I
in the prepara-
6B column,
Thus the molecular 100,000
was about
preparation
was
if
the
The
13-fold
that
of
colorless,
almost
any cytochrome.
to increase
weight
and 200,000.
preparation
little
the highest
More effec-
yield.
ACKNOWLEDGEMENTS The authors Science
Institute,
bacterial
strain
Ltd.
for
sistance
large
are grateful Machida used. scale
of Misses
to Dr. City,
They also culture
Toshiko
Tairo
Tokyo, thank
Oshima of Mitsubishi
for
kindly
Keiko
is acknowledged.
670
the
members of Kyowa Hakko Co.,
of the bacterium. Kanbe,
providing
Life
Ikeba
The technical and Machiko
as-
Yoshimura
Vol. 69, No. 3, 1976
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
REFERENCES 1.
2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
Kaback, H. R. (1974) Science 186, 882-892 Mitchell, P. (1966) Biol. Rev.41, 445-502 Harold, F. M. (1974) Ann. N. Y. Acad. Sci. 227, 297-311 Hirata, H., Altendorf, K., and Harold, F. M. (1974) J. Biol. Chem. 249, 2939-2945 Altendorf, K., Hirata, H., and Harold, F. M. (1975) J. Biol. Chem. 250, 1405-1412 Yoshida, M., Sone, N., Hirata, H., and Kagawa, Y. (1975) J. Biol. Chem. 250, 7910-7916 Sone, N., Yoshida, M., Hirata, H., and Kagawa, Y. (1975) J. Biol. Chem. 250, 7917-7923 Kagawa, Y., and Racker, E. (1971) J. Biol. Chem. 246, 5477-5487 Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 Oshima, T., and Imahori, K. (1974) International J. of Systematic Bacteriology 24, 102-112 Kagawa, Y., Sone, N.,%irata, H., Yoshida, M., and Okamoto, H. in preparation
671